|
| Status |
Public on Mar 23, 2010 |
| Title |
Spneumoniae_genomicDNA_3 |
| Sample type |
genomic |
| |
|
| Source name |
S. pneumoniae cell culture
|
| Organism |
Streptococcus pneumoniae |
| Characteristics |
strain: TIGR4
|
| Biomaterial provider |
ATCC
|
| Growth protocol |
Grown statically at 37ºC in Todd Hewitt broth. Cells were grown until the late-exponential phase of growth (OD600~1.5–2)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation and purification using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s specifications
|
| Label |
Cy3
|
| Label protocol |
Each DNA aliquot was fragmented by sonication to obtain fragments from 400 to 1000bp. Fragmented DNA was mixed with 5 µl 10X NEBlot labelling buffer containing random sequence octamer oligonucleotides (New England Biolabs, Ipswich, MA, USA) and water to a final volume of 43.5 µl. This mixture was denatured by heating to 95ºC for 5 min and then cooled down for 5 min at 4ºC. After this denaturing step, the remaining components of the labelling reaction were added: 5 µl of 10 X dNTP labelling mix (1.2 mM each dATP, dGTP and dCTP in 10 mM Tris pH 8.0, 1 mM EDTA), 1.5 µL of 1 mM Cy3-dUTP or Cy5-dUTP and 1.5 µl of 10 U/µl Klenow fragment (Fermentas Life Sciences, Glen Burnie, MD, USA). The labelling reactions were incubated overnight at 37ºC and then stopped by adding 2.5 µL of 0.5 M EDTA
|
| |
|
| Hybridization protocol |
Labelled samples were mixed in 45 µl EGT hybridization solution (Eurogentec, Serain, Belgium) and denatured at 65ºC for 2 min. The hybridization mixture was then loaded onto a microarray slide, covered with a coverslip and incubated at 38ºC overnight
|
| Scan protocol |
Scanning was perfomed using a Scanarray HT microarray scanner (Perkin-Elmer).
Scanned images were quantified using Imagene 6.0 software.
|
| Description |
Experiment purpose is identifying spots with positive signal
|
| Data processing |
VALUE was obtained as the background corrected data using the following formula "VALUE=SIGNAL_RAW-(BKD_RAW-2*(BKDstd)"
|
| |
|
| Submission date |
Oct 13, 2009 |
| Last update date |
Mar 22, 2010 |
| Contact name |
Guillermo López-Campos |
| Organization name |
Queen's University of Belfast
|
| Department |
Centre for Experimental Medicine
|
| Street address |
97 Lisburn road
|
| City |
Belfast |
| State/province |
Northern Ireland |
| ZIP/Postal code |
BT9 7BL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL5781 |
| Series (1) |
| GSE19005 |
Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization |
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